Fusion proteins for the treatment of cns

ABSTRACT

This disclosure relates to compositions capable of use in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS), and in particular, compositions including proteoglycan degrading molecules and compositions capable of blocking and/or over coming the activity of neuronal growth inhibitory molecules, as well as fusion proteins which includes a proteoglycan degrading domain and a domain capable of blocking and or over coming the activity of neuronal growth inhibitory molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of pending U.S.application Ser. No. 10/848,564, filed May 17, 2004, which claims thebenefit and priority of U.S. Provisional Application No. 60/471,239,filed May 16, 2003, U.S. Provisional Application No. 60/471,240, filedMay 16, 2003, U.S. Provisional Application No. 60/471,300, filed May 16,2003 and U.S. Provisional Application No. 60/474,372, filed May 29,2003. These applications are hereby incorporated by reference in theirentirety.

BACKGROUND AND SUMMARY

Spinal cord injury (SCI) inflicts trauma to the cells and tissues of thecentral nervous system (CNS) and causes a severe and debilitatingcondition in the individual. Following SCI, limited regeneration ofinjured neurons results in permanent disability characterized by someloss of sensation, paralysis and autonomic dysfunction. One reason thatneurons fail to regenerate is their inability to traverse the glial scarthat develops following SCI. This glial scar contains extracellularmatrix molecules including chondroitin sulfate proteoglycans (CSPGs). Invitro studies show that neurons fail to extend processes over CSPGcoated surfaces, while in vivo data correlate failure of regenerationwith areas of CSPG expression. Within the adult central nervous system(CNS) myelin there are also several identified axon growth inhibitorcompounds like myelin associated glycoprotein (MAG), OMgp, and Reticulon4 or Nogo that have been shown to be inhibitory for the growth ofneurons.

The proteglycan degrading enzyme chondroitinase ABC type I has been usedto enhance neuronal growth in a dorsal column lesion model of spinalcord injury. It has also been reported that treating a spinal cordinjury with NOGO receptor antagonist promotes a certain limited degreeof neuronal regeneration. It has further been reported that creating aNOGO knock out mouse resulted in certain limited and inconsistentdegrees of neuronal regeneration following dorsal hemisection of thespinal cord.

Experimental treatments for injury to the CNS have utilized theapplication of chondroitinase to the extracellular space, however theenzyme digests CSPG in the extracellular matrix and not intracellularstores. Furthermore, diffusion of chondroitinase within the parenchymathe essential and distinctive tissue of an organ or an abnormal growthas distinguished from its supportive framework) or between anatomicalcompartments is limited. The limited access of drugs, imaging agents,and anesthetics, etc. to target cells and/or tissues of the CentralNervous System (CNS) may reduce the usefulness or effectiveness of anyof these substances.

The delivery of therapeutic and diagnostic molecules into cells andtissues is, in part, dependent upon extracellular matrices as well ascarbohydrates and proteins linked to cell membranes. The extracellularmatrix is composed in part of proteoglycans, among them are thechondroitin sulfated proteoglycans (CSPGs). CSPGs are a family ofproteoglycans composed of a core protein and covalently linked sulfatedglycosaminoglycans. Each proteoglycan is determined by theglycosaminoglycan side chains. For CSPGs these side chains are made upof approximately 40 to 100 sulfated disaccharides composed ofchondroitin 4, 6 and dermatan sulfates. The protein component of theCSPG is ribosomally synthesized and the glycosylation occurs in theendoplasmic reticulum and Golgi apparatus. The sugar chains are thensulfated at the 4 or 6 positions by several glycosaminoglycansulfotransferases.

Transduction proteins may be used to transport polypeptides andpolynucleotides cargo across anatomical barriers and into cells. Forexample, the TAT protein (SEQ ID NO: 2) from the human immunodeficiencyvirus (HIV) contains a protein transduction domain (PTD) that isinvolved in the transduction of HIV into cells. The PTD contains an 11amino acid domain (TAT Peptide) (SEQ ID NO: 3) that is responsible forthe activity of the PTD. The TAT Peptide (SEQ ID NO: 3) can be linked toproteins and facilitate the transduction of the proteins into cells. Themechanism of transduction is independent of the molecular weight orchemical properties of the proteins that are linked to the TAT Peptide(SEQ ID NO: 3). In vivo studies show that if a fusion protein consistingof the TAT Peptide (SEQ ID NO: 3) linked to the 120 kd enzyme,beta-galactosidase β-Gal), is injected into mice, then a robust deliveryof β-Gal into a wide variety of cells is observed. When linked to theTAT transduction peptide (SEQ ID NO: 3), β-Gal activity was observed inthe brain; without the TAT Peptide (SEQ ID NO: 3), β-Gal was notobserved in the brain. Transport across the blood brain barrier isnormally restricted to certain hydrophobic small molecules andparticular low molecular weight lipophilic peptides. Transport ofproteins as large as β-Gal into the brain is usually not possiblewithout substantial disruption of the blood brain barrier, but the TATPeptide (SEQ ID NO: 3) facilitates transport while leaving the bloodbrain barrier intact.

Chimeric proteins, also called fusion proteins, are hybrid proteinswhich combine at least parts of two or more precursor proteins orpolypeptides. Chimeric proteins may be produced by recombinanttechnology, i.e. by fusing at least a part of the coding sequence of onegene to at least a part of the coding sequence of another gene. Thefused gene may then be used to transform a suitable organism which thenexpresses the fusion protein.

Tat Peptide Complexes Frankel et al. (U.S. Pat. Nos. 5,804,604;5,747,641; 5,674,980; 5,670,617; 5,652,122) discloses the use of Tatpeptides to transport covalently linked biologically active cargomolecules into the cytoplasm and nuclei of cells. Frankel only disclosescovalently linked cargo moieties that are (therapeutic, diagnostic orprophylactic), and does not teach or suggest the attachment of moleculesthat facilitate diffusion, plasticity, neurite growth, and axonregeneration. These molecules can include but are not limited tomolecules that overcome neurite out growth inhibition, or promote nervegrowth such as soluble NOGO antagonists like NgR₂₇₋₃₁₁, neural celladhesion molecules like L1, neurotrophic factors, growth factors,phosphodiesterase inhibitors, and inhibitors of MAG or MOG.Additionally, deletion mutants may be combined with other compounds thatpromote remyelination such as neuregulins (GGF2) and antibodies thatpromote remyelination or proteoglycan degrading molecules to Tatpeptides.

Regeneration following SCI is limited because of a variety of obstaclesthat include the deposition of CSPG at the glial scar, demyelination ofaxons, lack of trophic support and lack of axonal guidance. A singletherapy directed against one aspect of SCI may not be as effective as acombinatorial approach. Fusion proteins with chondroitinase will allowcombinatorial therapy with a single protein. Fusion partners forchondroitinase that will be constructed in this proposal were chosenfrom among proteins that have evidence for efficacy in SCI.

The use of a molecule that has the ability to both degrade extracellularmatrix glycoproteins and to block or overcome the inhibitory nature ofmyelin components may be used to improve the ability of damaged neuronsto grow or regenerate compared with either treatment alone. Theproteoglycan degrading molecules may also be used advantageously toprovide a method of facilitating access and diffusion of substances intocells or tissues through the use of at least one enzyme capable ofcleaving proteoglycans and preferably degrading chondroitin sulfateproteoglycans (CSPG).

Embodiments of the present invention include compositions that comprisepolypeptides which cleave proteoglycans, polypeptides that block and/orovercome the activity of neuronal growth inhibitory molecules, or acombination of these. The compositions containing the proteoglycandegrading molecule or neuronal growth inhibitory molecules may alsoinclude molecules for transduction of the polypeptides across cellmembranes and the blood brain barrier. The compositions may be used inthe treatment of spinal cord injuries and related disorders of thecentral nervous system (CNS). The compositions can be used in theregeneration of damaged neurological tissue and facilitate the diffusionand transport of therapeutic molecules capable of blocking and/orovercoming the activity of neuronal growth inhibitory molecules intodamaged or diseased tissue.

Embodiments of the present invention include compositions and methodsfor their use to facilitate delivery and diffusion of therapeutics ordiagnostic agents, and preferably agents that promote regeneration ofnerves and axons, into cells or tissues. Preferably the compositionincludes the use of an enzyme capable of cleaving chondroitin sulfateproteoglycans (CSPG) to increase the diffusion of these agents intocells or tissues of the central nervous system.

Compositions of the present invention may include chimeric or fusionproteins capable of systemic use in the treatment of spinal cordinjuries and related disorders of the central nervous system (CNS), andin particular, fusion proteins capable of crossing the blood brainbarrier. The fusion protein may include a polypeptide transductiondomain, a polypeptide domain capable of degrading a proteoglycan,preferably a domain cleaving chondroitin sulfate proteoglycan (CSPG), apolypeptide domain that blocks and or over comes the activity ofneuronal growth inhibitory molecules, or any combination of thesepolypeptide domains that may be used in the treatment of spinal cordinjuries and related disorders of the central nervous system (CNS). Thevarious polypeptide domains may be linked or chemically bonded togetherby polypeptide linkers.

Compositions of the present invention include polynucleotides whichencode for the chimeric or fusion proteins capable of systemic use inthe treatment of spinal cord injuries and related disorders of thecentral nervous system (CNS), and in particular, they encode for fusionproteins capable of crossing the blood brain barrier. Thepolynucleotides which encode for these chimeric or fusion proteins mayinclude a polynucleotide domain encoding for a polypeptide transductiondomain, a polynucleotide domain encoding for a polypeptide domaincapable of degrading a proteoglycan, preferably cleaving chondroitinsulfate proteoglycan (CSPG), a polynucleotide domain encoding for apolypeptide domain that blocks and or over comes the activity ofneuronal growth inhibitory molecules, or any combination of thesedomains that may be used in the treatment of spinal cord injuries andrelated disorders of the central nervous system (CNS). Thepolynucleotide also includes one or more polynucleotide domains thatencode for polypeptides that link the domains of the polypeptidetogether to form the fusion protein.

One embodiment of the present invention is a composition and a methodfor its use that facilitates the access and distribution of therapeuticand diagnostic agent in the composition into cells, through membranes orinto tissues by the use of composition that includes at least one enzymecapable of cleaving proteoglycans, preferably the composition includes afusion protein having an enzyme capable of cleaving CSPGs. The moleculesor agents in the composition may include one or more of Growth factorsincluding, Brain Derived Neurotrophic Factor, Insulin-like GrowthFactor, Fibroblast Growth Factor, Ciliary Neurotrophic Factor, GlialDerived Neurotrophic Factor, Transforming Growth Factor, Glial GrowthFactor 2, L1, GM1, Vascular Endothelial Growth Factor, Nerve GrowthFactor, Immunophilins. Molecules in the composition can includefluorescent or contrast agents for imaging. The agents may include cellsfor transplant—stem cells, neurons, others, cells as delivery agents,chemotherapeutic agents, antibiotics, antibody therapies, Nogo receptorantagonists, other chondroitinase enzymes. The composition may include atransduction domain, an enzyme capable of cleaving proteoglycans, orboth. Preferably the composition includes a fusion protein having atransduction domain, an enzyme domain capable of cleaving proteoglycans,or both. The fusion protein can facilitate the transport or modifiestransport of such agents into cells, tissues, and/or otherwiseinaccessible locations; and/or to enhance penetration rates, distance ofpenetration; or provide more even concentration distribution. Preferablythe modified transport occurs through the use of at least one enzymecapable of cleaving CSPGs. The compositions can be used for treating aCNS injury, preferably the composition is used in the treatment ofneuronal damage from a contusion injury.

Embodiments of the present invention include chimeric proteins of aproteoglycan degrading domain linked to a polypeptide that blocks theaction of neuronal growth inhibitors such as but not limited to aNogo-receptor antagonist (NgR₂₇₋₃₁₁) domain or variant linked to achondroitinase like chondroitinase ABC I or a variant of chondroitinasehaving one or more N terminal amino acids deleted. The compound mayinclude chimeric proteins of a proteoglycan degrading domain linked to apolypeptide that is a neural cell adhesion promoter such as an L1 neuralcell adhesion promoter domain or variant linked to chondroitinase ABC Ior a variant of chondroitinase having one or more N terminal amino acidsdeleted. The chimeric proteins may include chimeric proteins of aproteoglycan degrading domain linked to a polypeptide that is a glialcell stimulator, such as but not limited to a GGF2 glial cell stimulatoror variant linked to chondroitinase ABCI or a variant of chondroitinasehaving one or more N terminal amino acids deleted.

An E. Coli recombinant expression system and purification process can beused to produce essentially pure and catalytically active chondroitinaseABCI. These methods may be modified for producing chimeras ofproteoglycan degrading molecules and other agents.

The chimera may be assayed for chondroitinase enzymatic activity and thespecific biological activity of each fusion partner. Methods to measurethe activities of the chimera may be modified for those used to measurechondroitinase activity including a spectrophotometric assay,zymography, an HPLC assay to detect CSPG disaccharide digestion productsand an in vito neurite outgrowth assay. A neuron growth cone collapseassay can be used to evaluate NOGO receptor antagonists and a neuriteoutgrowth assay can be used measure L1 activity. GGF2 activity may bemeasured using a Schwann cell proliferation assay.

The compositions and method of the present invention can be used for thetreatment of spinal cord injuries and in the promotion of regenerationof axons. The compositions of the present invention can also be used topromote plasticity, regrowth, repair, and/or regeneration ofdysfunctional neurons in the CNS that have been damaged as a result ofdisease, such as degenerative diseases including Alzheimer's andParkinson's disease. Advantageously, the use of proteoglycan degradingpolypeptides or membrane transducing polypeptides in the compositions ofthe present invention also promote diffusion and access of damage ordiseased tissue to other therapeutic agents promoting the regenerationof neurons.

DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing/photographexecuted in color. Copies of this patent with colordrawing(s)/photograph(s) will be provided by the Office upon request andpayment of the necessary fee.

In part, other aspects, features, benefits and advantages of theembodiments of the present invention will be apparent with regard to thefollowing description, appended claims and accompanying drawings where:

FIG. 1 is an illustrative example of a TAT-chondroitinase ABCIconstruct; the DNA sequence for the entire gene fragment fused with theTat sequence followed by the 5-glycine linker (SEQ ID NO: 45); the senseand antisense oligonucleotides having the sequences as 5′-tatgtatggtcgtaaaaagcgtc gtcaacgtcgtcgtgg tggtggtggtca-3′ (SEQ ID NO: 92) and5′-tatgaccaccaccaccaccacgac gacgttgacgac gcttttt acgaccataca-3′ (SEQ IDNO: 93) were annealed and ligated at the NdeI site which is at the 5′end [f]of ABCI gene cloned in pET15b (Novagen) at the NdeI and BamHIsites.

FIG. 2 are Brain section images; (I) illustrating lobes from adult ratwhich were incubated in beta-galactodidase alone (B&D), or with theaddition of Choindroitinase ABCI (A, 0.5 U/ml or C, 0.005 U/ml); (II)Eosin Y penetration through the cortex of a Choindroitinase treatedbrain hemisphere and control showing approximately the same penetration,Eosin Y is a is a zwitterionic, having an overall negative charge at thelow pH it was used at, and it is 692 kDa; (III)A saturate solution ofCongo Red demonstrates greater penetration through the cortex of aChondroitinase treated brain hemisphere as compared to untreated brain,Congo red is a negatively charged dye of 697 kDa;

FIG. 3 (A) is a diagram representing the full length GGF protein(GGF2-M₁-E₄₂₂) and the three GGF2 fragments containing the Ig and EGFdomains GGF2-L₂₅₀-C₄₀₂, Ig and EGF domains GGF2-L₂₅₀-E₄₂₂ to C-terminal;and EGF domains GGF2-T₃₅₀-C₄₀₂; (B) a schematic presentation of chimericproteins of a proteoglycan degrading molecule like chondroitinase ABCIand a neuregulin 1 gene isoform GGF2 such as N-terminal fusion chimerabetween chondroitinase ABCI-NΔ60-CΔ80 and GGF2-FL, betweenchondroitinase ABCI-NΔ60-CΔ80 and GGF2-L₂₅₀-C₄₀₂, between chondroitinaseABCI-NΔ60-CΔ80 and GGF2-L₂₅₀-E₄₂₂, and between chondroitinaseABCI-NΔ60-CΔ80 and GGF2-T₃₅₀-C₄₀₂, and C-terminal fusion chimera (lower)such as between chondroitinase ABCI-FL and GGF2-FL, betweenchondroitinase ABCI-FL and GGF2-L₂₅₀-C₄₀₂, between chondroitinaseABCI-FL and GGF2-L₂₅₀-E₄₂₂, and between chondroitinase ABCI-FL andGGF2-T₃₅₀-C₄₀₂.

FIG. 4 illustrate the structure of (A) an NgR₂₇₋₃₁₁—N terminalchondroitinase ABCI chimeric protein without, and with, a peptide spaceror linking group; (B) an NgR₂₇₋₃₁₁—C terminal chondroitinase ABCIchimeric protein without, and with, a peptide spacer or linking group;(C) an extracellular domain L1 N-terminal chondroitinase ABCI chimericprotein without, and with, a spacer or linking peptide; (D) anextracellular domain L1 C-terminal chondroitinase ABCI chimeric proteinwithout, and with, a spacer or linking peptide.

FIG. 5 (A)BBB scores from spinal cord injured animals treated withchondroitinase (Chondroitinase ABC I), penicillinase or artificialcerebrospinal fluid (aCSF); (B) Parasagittal sections of spinal cordsfrom injured animals treated with chondroitinase (left column) orpenicillinase (right column). Sections were cut at 30 microns. Each setof images contains four parasagittal sections realigned with the rostralcord to the left and the caudal cord to the right. The most centralsection of each set has been removed and replaced below to aidvisualization. Pairs of images are Weil stained, anti-GFAP and an aminocupric stain of neuronal degeneration (top to bottom). (C) and (D)Distribution of individual BBB scores according to treatment group.Scores are BBB scores at ten weeks post surgery. Group averages areshown below the data points.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event occurs and instances where it does not.

Suitable enzymes that cleave CSPGs include chondroitinase ABC type I,chondroitinase ABC Type II, chondroitinase AC, and chondroitinase B, ormammalian enzymes with chondroitinase-like activity such ashyaluronidase-1, hyaluronidase 2, hyaluronidase 3, hyaluronidase 4,cathepsins, ADAMTs and PH-20, or mixtures thereof.

CSPGs are a family of proteoglycans composed of a core protein andcovalently linked sulfated glycosaminoglycans. The polysaccharide chainsare cleaved by several enzymes, including a family of chondroitinases.To date, this enzyme family contains at least four members,Chondroitinase ABC I, ABC II, AC and B. Chondroitinase ABC I is anexo-lyase which cleaves both chondroitin and dermatan sulfates. It hasbeen used extensively in the study of in vitro neuronal regenerationover CSPG-laden substrates and more recently in in vivo studiesfollowing CNS injury.

Proteins and polypeptide that may be used in the compositions and fusionproteins of the present invention are those which promote plasticity aswell as the regeneration of injured or diseased neurons and axons. Theseregenerating proteins and polypeptides may include cell adhesionproteins, those which stimulate glial cells, and polypeptides whichblock the inhibitory effect of proteins that act as axon growthinhibitors.

Plasticity of the nervous system refers to any type of functionalreorganization. This reorganization occurs with development, learningand memory and brain repair. The structural changes that occur withplasticity may include synapse formation, synapse removal, neuritesprouting and may even include strengthening or weakening existingsynapses. Regeneration is generally differentiated from plasticity bythe long range growth of axons in disrupted tracts that ischaracteristic of regeneration.

Proteins and polypeptides are capable of blocking the activity ofneuronal growth inhibitory molecules may include peptides andpolypeptides that block the inhibitory properties of peptide such as butnot limited Nogo, MAG, and OMgp. Suitable compositions that overcome theactivity of neuronal growth inhibitory molecules include but are notlimited to Protein Kinase C family inhibitors, Rho Kinase familyinhibitors and agents, such as phosphodiesterase inhibitors, thatincrease intracellular cyclic AMP, and L1. The (NgR₂₇₋₃₁₁) peptide hasbeen shown to inhibit binding of Nogo66, OMgp, MAG, and MOG tomembrane-bound NogoR and overcome the inhibitory effects of Nogo on theregeneration of nerve processes.

Proteins and polypeptides that affect cell adhesion or stimulate cellsmay include but are not limited to poly peptides such as L1 and GGF2. L1is a neural cell adhesion protein that is a potent stimulator of neuriteoutgrowth in vitro for which has been found that treatment of acute SCIin rodents using a soluble form of L1 leads to an increase in therecovery of neurological function. GGF2 stimulates glial cells and hasbeen shown to improve clinical outcome measures in a murine model ofexperimental allergic encephalomyelitis (EAE) likely as a result of thestimulation of oligodendrocytes to promote remyelination.

Cell membrane-permeant peptide sequences useful in practicing thepresent invention include, but are not limited to, RQARRNRRRRWRERQR-51(HIV-1 Rev protein basic motif; (SEQ ID NO:43)); MPKTRRRPRRSQRKRPPTP-119(HTLV-1 Rex protein basic motif; (SEQ ID NO:44)) (Kubota et al. 1989);the third helix of the homeodomain of Antennapedia (Derossi, et al., J.Biol. Chem. 271:18188-93, 1996) (43-RQIKIWFQNRRMKWKK-58 (SEQ ID NO:45));a peptide derivable from the heavy chain variable region of an anti-DNAmonoclonal antibody (Avrameas, et al., Proc. Natl. Acad. Sci.95:5601-06, 1998) (VAYISRGGVSTYYSDTVKGRFTRQKYNKRA (SEQ ID NO:46)); andthe Herpes simplex virus VP22 protein (Elliot and O'Hare, Cell,88:223-33, 1997) (1-MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASRAPTVQLWQMSRPRTDEDLNELLGITHRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE-246 (SEQ ID NO:47)). In apreferred embodiment, the basic peptide is derivable from the humanimmunodeficiency virus type 1 (HIV-1) Tat protein (Fawell et al., Proc.Natl. Acad. Sci., 91:664-68, 1994). In particular, the Tat peptide cancomprise any sequential residues of the Tat protein basic peptide motif37-72 (Vives et al. 1997) (37-CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ-72(SEQ ID NO:48)). The minimum number of amino acid residues can be in therange of from about three to about six, preferably from about three toabout five, and most preferably about four, i.e., the minimalrequirement for one alpha helical turn. A preferred embodiment comprisesTat protein residues 48-57 (GRKKRRQRRR) (SEQ ID NO:49).

Suitable PTD domains include those derived from the TAT protein (SEQ IDNO: 2). Tat proteins and peptides: Tat (SEQ ID NO: 2) is an 86-aminoacid protein involved in the replication of human immunodeficiency virustype 1 (HIV-1). The HIV-1 Tat transactivation protein (SEQ ID NO: 2) isefficiently taken up by cells (Mann and Frankel 1991; Vives et al.1994), and low concentrations (nM) are sufficient to transactivate areporter gene expressed from the HIV-1 promoter (Mann and Frankel 1991).Exogenous Tat protein (SEQ ID NO: 2) is able to translocate through theplasma membrane and reach the nucleus to transactivate the viral genome.

A region of the Tat protein (SEQ ID NO: 2) centered on a cluster ofbasic amino acids is believed to be responsible for this translocationactivity (Vives et al. 1997). Tat peptide-mediated cellular uptake andnuclear translocation have been demonstrated in several systems (Vives,et al., J Biol Chem 272:16010-16017, 1997; Jones, Genes Dev11:2593-2599, 1997). Chemically coupling a Tat-derived peptide (residues37-72) (SEQ ID NO:48) to several proteins results in theirinternalization in several cell lines or tissues (Fawell, et al., ProcNatl Acad Sci USA 91:664-668, 1994; Anderson, et al., Biochem BiophysRes Commun 194:876-8884, 1993; Fahraeus, et al., Curr Biol 6:84-91,1996; Nagahara, et al., Nat Med 4:1449-1452, 1998). A synthetic peptideconsisting of the Tat basic amino acids 48-60 with a cysteine residue atthe C-terminus coupled to fluorescein maleimide translocates to the cellnucleus as determined by fluorescence microscopy (Vives et al. 1997). Inaddition, a fusion protein (Tat-NLS-.beta.-Gal) consisting of Tat aminoacids 48-59 fused by their amino-terminus to .beta.-galactosidase aminoacids 9-1023 translocates to the cell nucleus in an ATP-dependent,cytosolic factor-independent manner (Efthymiadis et al. 1998).

Chimeric proteins, also referred to in the art as fusion proteins, arehybrid proteins which combine at least parts of two or more precursorproteins or peptides. Chimeric proteins may be produced by recombinanttechnology, i.e. by fusing at least a part of the coding sequence of onegene to at least a part of the coding sequence of another gene. Wheredesirable, one or more genes for linker peptides may be fused to thecoding sequence of genes for the other polypeptide domains in the fusionprotein. The fused gene may then be used to transform a suitableorganism such as but not limited to E. coli or CHO cells which thenexpresses the fusion protein.

Genes encoding either N or C terminal deletion mutants of polypeptidedomains of the fusion proteins can be used in constructs for expressionof the fusion proteins. Preferably the generated deletion mutantsmaintain their catalytic proteoglycan degrading activity, blockingactivity, growth activity, or transduction activity. Generated deletionmutants of the proteoglycan degrading molecules like chondroitinase ABCIenzyme where the mutant is missing a certain number of amino acids fromthe N and or C-terminal are those that retain some proteoglycandegrading activity. N-terminal deletions of chondriotinase likechondroitinase ABC I maintain a histidine-tag that is attached to theN-terminus. It is expected that a TAT-deletion mutant chondroitinase ABCI fusion DNA construct can be expressed without removal of the TATpolypeptide during expression. For example a TAT peptide can be fused atthe N-terminus of either ABCI-NΔ20 or ABCI-NΔ60 deletion mutant.Fragments of polypeptides, such as those shown in FIG. 3 for GGF2, maybe used in the construction of chimeric fusion proteins.

Catalytically active deletion mutants of chondroitinase ABCI can beprepared for example but not limited to deleting 20, 40 and 60 aminoacids respectively from the N-terminus of the mature ABCI protein.Deletion of 60 amino acids from the N terminal and 80 amino acids fromthe C-terminal end may also be used to make a deletion mutants of aproteoglycan degrading chondroitinase ABCI. These deletion mutants andthose of other proteoglycan degrading molecules may be used forconstruction of N-terminal fusion chimeric protein. Detailed comparativebiochemical studies can be done to determine the efficacy of maturechondroitinase ABCI versus various deletion mutant in compositions andfusion proteins with respect to the substrate specificity, substratebinding and tissue penetration.

A mutant of chondroitinase ABCI that has native protein structure, butlacks catalytic activity may be prepared as a null or a negative controlfor bioassays and SCI studies. Based on the crystal structure ofchondroitinase ABCI a site-specific mutant designated H501a and Y508a toknock out catalytic activity in the putative active site can beprepared. Such mutants can be tested for inactivation of catalyticactivity and SEC to compare to the wild-type enzyme. If the nullactivity mutant is successfully created it will provide a negativecontrol for the various fusion proteins for use in bioassays andultimately in SCI animal studies.

An E. coli expression system may be used to make chondroitinase usingPET expression vectors (Novagen). The GGF2-chondroitinase ABCI fusionprotein may be expressed in E. coli. Constructs for Tat-chondroitinasedeletion mutant fusion proteins, Tat-GGF2 fusion proteins, orTat-chondroitinase-GGF2 fusion proteins may be expressed from E. coli.Other fusion proteins can be expressed in CHO cell lines.

Table 1 illustrates various non-limiting components which may be used incompositions of the present invention as a mixture, and preferably as afusion or chimeric molecule. The composition or chimeric moleculedescribed may include one or more of the molecules in Table 1. In thecase of chimeric molecules one or more linker segments, preferablypolypeptides are used.

TABLE 1 Components of Compositions Proteoglycan degrading Therapeutic,diagnostic, Transduction molecules molecules receptor antagonistmolecules Tat protein residues 48-57 Any agent that degrades Any peptidethat blocks the (SEQ ID NO: 49) extracellular matrix inhibitoryproperties of Nogo, HIV-1 Rev protein basic glycoproteins including:MAG, OMgp including: motif (SEQ ID NO: 43) Chrondroitinase ABC I, Nogopeptide 1-40 HIV-1 Rev protein basic Chondroitinase ABC II, Anycomponent of Nogo motif(SEQ ID NO: 43) Chondroitinase AC, peptide 1-40that maintains the Herpes simplex virus Chondroitinase B, ability toblock the inhibitory VP22 protein Hyaluronidase 1, properties of NogoHyaluronidase 2, Other blocking peptides of Hyaluronidase 3, NogoHyaluronidase 4, Antibodies that recognize Nogo PH20 Blocking peptidesof MAG deletion and or substitution Antibodies that recognize MAGmutants of the above listed Blocking peptides of OMgp molecules thatmaintains Antibodies that recognize enzymatic activity. OMgp Antibodiesthat recognize the Nogo-66 receptor Blocking peptides of the p75receptor Antibodies that recognize the p75 neurotrophin receptorPeptides or antibodies that block other receptors of Nogo, MAG and/orOMgp Peptide that overcomes the inhibitory properties of Myelin, Nogo,MAG, OMgp including: Protein Kinase C family inhibitors* Rho Kinasefamily inhibitors Agents that increase intracellular cAMP concentrationL1

A schematic illustration of non-limiting versions of chimeric fusionproteins of the present invention are illustrated in FIG. 4A and FIG.4B.

A peptide component from any column in Table 1 could be linked by anoligopeptide linker that is well known in the art. A glycine richpeptide, for example Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 56), or linkersprepared including any of the naturally occurring amino acids as well assubstituted or beta or gamma amino acids like 4-aminobutyric acid or6-aminocaproic acid can be used. Other linkers including but not limitedto alkyl diamines, amino, or alkyl diols may also be used. Preferablythe transduction component of the fusion protein is in a terminalposition in the polypeptide. Other examples of common linkers mayinclude but would not be limited to Gly-Gly-Ala-Gly-Gly (SEQ ID NO: 57),Gly/Ser rich linkers (for example Gly₄Ser₃ (SEQ ID NO: 94)), or Gly/Alarich linkers. Additionally, linkers may be of any length and design topromote or restrict the mobility of components in the fusion protein.

The incorporation of non-natural amino acids, including syntheticnon-native amino acids, substituted amino acids, or one or more D-aminoacids into the peptides (or other components of the complexes) of thepresent invention (subsequently referred to herein as “D-peptides”) isadvantageous in a number of different ways. D-amino acid-containingpeptides exhibit increased stability in vitro or in vivo compared toL-amino acid-containing counterparts. Thus, the construction of peptidesincorporating D-amino acids can be particularly useful when greaterintracellular stability is desired or required. More specifically,D-peptides are resistant to endogenous peptidases and proteases, therebyproviding better oral transepithelial and transdermal delivery of linkeddrugs and conjugates, improved bioavailability of membrane-permeantcomplexes, and prolonged intravascular and interstitial lifetimes whensuch properties are desirable. The use of D-peptides can also enhancetransdermal and oral transepithelial delivery of linked drugs and othercargo molecules.

In a spinal cord injury, the axons of ascending sensory and descendingmotor neurons are disrupted, that can result in the loss of sensationand paralysis. These axons fail to regenerate successfully leading topermanent disability. A scar envelopes the site of the injury which isbelieved to wall off the area of fragile tissue, stabilize the bloodbrain barrier, and prevent an overwhelming cascade of uncontrolledtissue damage. This scar is composed of hypertrophic glial cells and anextracellular matrix (ECM). Chondroitin sulfate proteoglycans (CSPGs)are one important component of the scar. They are expressed by glialcells and deposited in the ECM in regions of blood brain barrierbreakdown. In vitro evidence demonstrates that these CSPGs are potentlyinhibitory for the growth of axons and without wishing to be bound bytheory, are believed to contribute to the failure of the spinal cordaxons to regenerate and reform functional synapses. In vivo studies havedemonstrated that regenerating axons are able to grow into and evenbeyond the scar.

CSPGs and white matter components are generally accepted as molecularbarriers that neurons must overcome in order to regenerate andreestablish functional connections after injury. Transplanted adultsensory neurons placed distal to a forming scar can regenerate robustlyeven along degenerating white matter pathways, however, regenerationceases abruptly as axons enter the proteoglycan containing glial scar.Treatment of CNS white matter pathways with chondroitinase enhances theability of neurons to grow in these substrates.

Central nervous system tissues are tightly compacted with cells and havelimited extracelluar space. The proteins and carbohydrates of theextracellular matrix provide charge and osmotic forces as well asspecific and non-specific binding sites which may prevent thepenetration of therapeutic agents. The enzymatic cleavage of thesematrix and cellular components may later or facilitate the access ofcompounds or cells through tissues. A proteoglycan degrading moleculelike Chondroitinase ABC I that is an enzyme that digests chondroitinsulfate proteoglycans can be used to promote diffusion of therapeuticmolecules into the CNS. Tat peptides transport covalently linkedbiologically active cargo molecules into the cytoplasm and nuclei ofcells. In the case of a fusion protein having a protein transductionpolypeptide domain like the HIV tat protein (SEQ ID NO: 3), therapeuticmolecules for axon regeneration may be delivered across the blood brainbarrier.

Treatment of SCI model injuries, preferably contusion model injury, canbe used to determine the degree of regeneration and functional recoveryachieved by compositions and method of the present invention. The degreeof functional recovery can be demonstrated by improved corticospinaltract conduction, improved tape removal, beam walking, grid walking andpaw placement following chondroitinase treatment of a dorsal columnlesion. Motor skill improvement as well as autonomic function: bowel,bladder, sensor and sexual function may also be used as measures offunction improvement and related to molecular structure and componentsin the compositions of the present invention.

In addition to the ability of chondroitinase to enhance regeneration,chondroitinase digests components of the perineuronal network (PNN). Thedensity of the PNN is variable within the CNS and is particularly densein the somatosensory, auditory, visual cortices and the hippocampus. PNNhas also been demonstrated to be dense around spinal motor neurons. Theinventors have discovered the dense PNN within the dorsal horn of thecord. Digestion of the PNN can enhance plasticity within the hippocampusand visual cortex. Plasticity within intact systems in incomplete SCI,especially in the region of the central pattern generators or in thereticular core, may support the function of damaged or destroyedsystems. Promotion of plasticity in these systems may be one mechanismother than or in addition to regeneration by which chondroitinase canimprove function following CNS injury. Furthermore, regeneration andplasticity may work in concert to affect recovery following injury;indeed, the corticospinal tract has been shown to be critical formodulation of spinal cord plasticity.

Recovery of neurological function following contusion injury in the CNSor a disease state may be promoted by administering the fusion proteinsor mixtures including one or more of the components in Table 1, tocells, a tissue, or a subject having damaged or diseased neurons whetherthe injury or disease is immediate or long-standing.

The fusion proteins herein are administered in an amount effective todegrade CSPGs and thereby promote the recovery of neurological function.Once the proteins or polypeptides in the compositions have been purifiedto the extent desired, they may be suspended or diluted in anappropriate physiological carrier or excipient for SCI treatment. Inmodels of SCI, effective intrathecal doses in rats have been about 0.06units on alternate days for 14 days. A dose for a 70 kilogram human maybe about 17 Units. At about 100 Units/milligram, this would equal about170 micrograms. Doses of up to 20 Units appear safe in the rat.Compositions including a proteoglycan degrading molecule in a mixture oras part of a fusion protein diluted in a carrier or pharmaceuticallyacceptable excipient can be injected, generally at concentrations in therange of 1 μg to 500 mg/kg of host. Administering the agent can be bybolus injection, intravenous delivery, continuous infusion, sustainedrelease from implants, or sustained release pharmaceuticals.Administration may be by injection, such as intramuscularly,peritoneally, subcutaneously, intravenously. Oral administration mayinclude tablets or capsules, preferably the oral dosage is a sustainedrelease formulation for once or twice daily administration. Percutneousadministration can be once per day, and is preferably less than once perday administration. Administration to the human patient or othermammalian subject may be continued until a measurable improvement inautonomic or motor function in the patient is achieved.

The chondroitinase PTD fusion proteins can be administered with asuitable pharmaceutical carrier. The administration of the compositionsof the present invention as mixtures or chimeric proteins can betopical, local or systemic. The chimeric fusion proteins may also besecreted by genetically cells, preferably a chimeric fusion proteinhaving a proteoglycan degrading portion like chondroitinase and atransduction polypeptide portion like TAT can be secreted by geneticallymodified cells that are implanted, either free or in a capsule, at ornear the site of CNS injury.

Once the compositions either as mixtures or fusion proteins areadministered, degradation of CSPGs removes the inhibitory molecules thatblock neurite outgrowth, and allow the regeneration of neurites into theaffected area. For example, the chondroitinase AC and chondroitinase Bdegrade CS and DS, respectively, resulting in unsaturated sulfateddisaccharides. Chondroitinase AC cleaves CS at 1, 4 glycosidic linkagesbetween N-acetylgalactosamine and glucuronic acid in the polysaccharidebackbone of CS. Cleavage occurs through beta-elimination in a randomendolytic action pattern. Chondroitinase B cleaves the 1, 4galactosamine iduronic acid linkage in the polysaccharide backbone ofDS. The cleavage of both CS and DS occurs through a beta-eliminationprocess which differentiates these enzymatic mechanisms from mammalianGAG degrading enzymes. Chondroitinase ABCI, and chondroitinase ABCII,are exo and endo lyases that cleave both CS and DS. The removal of CSand DS from the glial scar permits the regeneration of neuriteoutgrowths into the injured area.

Mixtures of any of these fusion polypeptides may be used to provide atherapeutic treatment for CNS injuries and disorders which may includebut are not limited to contusion injury, traumatic brain injury, stroke,multiple sclerosis, brachial plexus injury, amblioplia, spinal cordinjuries. Spinal cord injuries includes disease and traumatic injuries,such as the crushing of neurons brought about by an auto accident, fall,contusion, or bullet wound, as well as other injuries. Practice of thepresent methods can confer clinical benefits to the treated mammal,providing clinically relevant improvements in at least one of thesubject's motor coordination functions and sensory perception.Clinically relevant improvements can range from a detectable improvementto a complete restoration of an impaired or lost function of the CNS.

The regeneration of the nerve cells into the affected CNS area allowsthe return of motor and sensory function. Clinically relevantimprovement will range from a detectable improvement to a completerestoration of an impaired or lost nervous function, varying with theindividual patients and injuries.

A variant of a protein or fragments thereof refer to a moleculesubstantially similar to either the entire protein or a fragment, whichpossesses biological activity that is substantially similar to abiological activity of the complement protein or fragments. A moleculeis substantially similar to another molecule if both molecules havesubstantially similar structures or if both molecules possess a similarbiological activity.

Variants of complement proteins or fragments thereof are produced bychemical or recombinant means. Variants of the polynucleotidesconstructed to express fusion proteins may also be made. The variantsmay include, for example, deletions from, or insertions or substitutionsof, amino acid residues within the amino acid sequence, or deletion,substitution, or insertion of nucleic acids from a sequence encoding fora particular fusion protein or polypeptide domain in the fusion protein.For example, in some cases the removal of one or more amino acidresidues from a chondroitinase polypeptide can be made withoutsignificant change in its CSPG degradation activity. Substantial changesin functional properties like proteoglycan degradation or blockingactivity against axon growth inhibitors are made by selectingsubstitutions that are less conservative, ie. that differ moresignificantly in their effect on maintaining the structure, charge orhydrophobicity of the peptide backbone in the area of the substitution.

Most deletions, insertions, and substitutions are not expected toproduce radical changes in the characteristics of the protein molecule;however, when it is difficult to predict the exact effect of thesubstitution, deletion, or insertion in advance of doing so, one skilledin the art will appreciate that the effect will be evaluated by routinescreening assays. For example, a change in the axon regenerationcharacter of the polypeptide molecule, through more or less proteoglycandegradation can be measured with functional recovery tests as well asHPLC assays to detect CSPG disaccharide digestion products.

The present invention relates to the use of a proteoglycan degradingmolecule, an HIV tat protein, or a tat-derived polypeptide, or acombination of these as a mixture or fusion protein to deliver amolecule of interest into the CNS or a site in the CNS where neuronaltissue damage has occurred by disease or trauma. In particular thedamage site in the CNS is where scaring has occurred as a result of acontusion injury. The molecule of interest, optionally referred to as anagent or cargo molecule can be a therapeutic molecule which promotesplasticity, axon growth, a diagnostic molecule, or a proteoglycandegrading molecule. In the case of a fusion protein having a proteintransduction polypeptide domain like the HIV tat protein (SEQ ID NO: 3),therapeutic molecules for axon regeneration may be delivered across theblood brain barrier or proteoglycan degrading molecule can be deliveredinto cells to degrade cellular stores of proteoclycans and promote axonregeneration.

Transport polypeptides of this invention may be advantageously attachedto cargo molecules by chemical cross-linking or by genetic fusion. Aunique terminal cysteine residue is a preferred means of chemicalcross-linking According to some preferred embodiments of this invention,the carboxy terminus of the transport moiety is genetically fused to theamino terminus of the cargo moiety. Embodiment of the present inventionconsists of an amino-terminal methionine followed by tat residues 47-58(SEQ ID NO: 49), followed by a chondroitinase polypeptide.

It will be appreciated that the entire 86 amino acids which make up thetat protein may not be required for the uptake activity of tat. Forexample, a protein fragment or a peptide which has fewer than the 86amino acids, but which exhibits uptake into cells and or can cross theblood brain barrier, can be used (a functionally effective fragment orportion of tat). For example, tat protein containing residues 1-72 canbe sufficient for uptake activity and tat residues 1-67 can mediate theentry of a heterologous protein into cells. Synthetic peptide containingtat residues 1-58 can have uptake activity.

The tat peptide can be a single (i.e., continuous) amino acid sequencepresent in tat protein or it can be two or more amino acid sequenceswhich are present in tat protein, but in the naturally-occurring proteinare separated by other amino acid sequences. As used herein, tat proteinincludes a naturally-occurring amino acid sequence which is the same asthat of naturally-occurring tat protein, its functional equivalent orfunctionally equivalent fragments thereof (peptides). Such functionalequivalents or functionally equivalent fragments can possess uptakeactivity into the cell or across the blood brain barrier that issubstantially similar to that of naturally-occurring tat protein. Tatprotein can be obtained from naturally-occurring sources or can beproduced using genetic engineering techniques or chemical synthesis.

The amino acid sequence of naturally-occurring HIV tat protein (SEQ IDNO: 2) can be modified, by addition, deletion and/or substitution of atleast one amino acid present in the naturally-occurring tat protein, toproduce modified tat protein (also referred to herein as tat protein orpolypeptide). Modified tat protein or tat peptide analogs with increasedstability can thus be produced using known techniques. Therefore, tatproteins or peptides may have amino acid sequences which aresubstantially similar, although not identical, to that ofnaturally-occurring tat protein or portions thereof. In addition,cholesterol or other lipid derivatives can be added to tat protein toproduce a modified tat having increased membrane solubility.

Naturally-occurring HIV-1 tat protein (SEQ ID NO: 2) has a region (aminoacids 22-37) wherein 7 out of 16 amino acids are cysteine. Thosecysteine residues are capable of forming disulfide bonds with eachother, with cysteine residues in the cysteine-rich region of other tatprotein molecules and with cysteine residues in a cargo protein or thecargo moiety of a conjugate. Such disulfide bond formation can causeloss of the cargo's biological activity. Furthermore, even if there isno potential for disulfide bonding to the cargo moiety (for example,when the cargo protein has no cysteine residues), disulfide bondformation between transport polypeptides can lead to aggregation andinsolubility of the transport polypeptide, the transportpolypeptide-cargo conjugate, or both. The tat cysteine-rich region maybe deleted to avoid disulfide bond formation and prevent aggregation andinsolubility of the transport polypeptide, the transportpolypeptide-cargo conjugate, or both.

Chondroitinase is also able to promote plasticity in regions of the CNSwith significantly dense PNN, including cortex, tectum, hippocampus andspinal cord. It is reasonable that some combination of effects,including regeneration, sprouting and plasticity are responsible for theimprovement in function following SCI with chondroitinase treatment ortreatment with fusion molecules including chondroitinase or otherproteoglycan degrading molecule.

NbR₂₇₋₃₁₁: Nogo is a high molecular weight myelin component thatinhibits neurite outgrowth. The amino terminal region (Nogo66) is thepart of the molecule that is specifically associated with the inhibitionof neurite outgrowth. Expression cloning methods revealed the receptorfor Nogo66 (NgR) is a GPI anchored glycoprotein expressed mainly onneurons. NgR interacts not only with Nogo, but also with other myelinassociated inhibitors such as MAG and MOG. Due to its central role inthe inhibitory properties of myelin on neurons, NgR has been the targetfor approaches to antagonize its interactions with its ligands. Thesoluble segment of NgR interacts with Nogo66, MAG and MOG. The regionspans residues 27-311 and this NgR fragment can therefore act as a decoyreceptor that will interfere with myelin associated inhibition ofneurons. If NgR₂₇₋₃₁₁ is linked to proteoglycan degrading molecule likechondroitinase ABCI to form a chimeric fusion protein, the NgR₂₇₋₃₁₁polypeptide domain of the fusion protein would be expect to limit themyelin-associated inhibition of neurite outgrowth and promote axonalregeneration in chondroitinase digested regions of a spinal cord injury.

For cloned (NgR₂₇₋₃₁₁), the precise region of interest, or fragments canbe derived from the initial clone. The biological activity of NgR₂₇₋₃₁₁,its fragments, and fusion polypeptides including NgR₂₇₋₃₁₁ may beconfirmed using an in vitro assay for the collapse of growth cones onneurons. The growth cone collapse assay has been established and theaddition of MAG or Nogo66 causes the collapse of growth cones in a dosedependent manner. If NgR₂₇₋₃₁₁, its fragments, and fusion polypeptidesincluding NgR₂₇₋₃₁₁ are active is biologically active then they shouldinhibit the MAG and Nogo66 mediated growth cone collapse. Growth conecollapse data may be collected by the inspection of photomicrographs ofDRG neurons in response to Nogo66 and MAG.

The L1 polypeptide is a member of the immunoglobulin superfamily of celladhesion molecules and is expressed in growing axons, glial progenitorcells and Schwann cells throughout life, but has only limited expressionin the CNS. L1 interacts with itself and with other extracellularmolecules, such as the FGF receptor to promote neurite fasciculation andgrowth. Expression of L1 is generally associated with a permissiveenvironment for axonal regeneration the, precise region of interest, orfragments of L1 can be derived from the initial clone. For example,Schwann cells that express L1 support peripheral nerve regeneration andaxonal growth is observed in the optic nerves from transgenic mice thatexpress L1 in astrocytes but not in wild-type optic nerves. Fibroblastsengineered to express L1 support axonal growth when transplanted intospinal cord. Finally, a soluble form of L1 linked to Fc promotedfunctional recovery following acute SCI. HU is linked to proteoglycandegrading molecule like chondroitinase ABCI in a fusion protein it isreasonable to expect the fusion protein to promote axonal regenerationin chondroitinase digested regions of a spinal cord injury.

The neuregulins and their receptors comprise a diverse growth factor andreceptor tyrosine kinase system that has been demonstrated to beessential for organogenesis in the CNS, muscle epithelial and othertissues. GGF2 is a soluble isoform of the neuregulin 1 gene. It wasinitially characterized as a Schwann cell mitogen 32, but subsequentstudies have demonstrated direct actions on oligodendrocytes, neuronsand other cell types. GGF2 diminishes demyelination and inflammation andenhances remyelination in a mouse model for multiple sclerosis. Based onthese results, it is reasonable to expect that a recombinant human GGF2is a potential treatment for demyelination associated with SCI. WithGGF2 is linked to a proteoglycan degrading molecule like chondroitinaseABCI it is reasonable to expect to promote remyelination of axons inchondroitinase-digested regions of a SCI.

TABLE 2 Examples of non-limiting GGF2 fragments for expression in E.coli for use in compositions and fusion polypeptide compositions.Nucleotide Amino Construct Domain Seq Acid Seq Mol. Wt. GGF2-FL Fulllength Ml-E422   46 kDa GGF2 GGF2/1 Ig domain + EGF 748-1206 L250-C40216.8 kDa domain GGF2/2 Ig domain to C- 748-1266 L250-E422   19 kDaterminus GGF2/3 EGF domain 1048-1206  T350-C402  5.7 kDa

The efficacy of a composition of the present invention, such as aproteoglycan degrading molecule and a molecule that blocks the activityof an axon growth inhibitor either as a mixture [of] or as a fusionprotein can be evaluated using a validated rat SCI model at threedifferent levels of SCI severity.

A proteoglycan degrading molecule like Chondroitinase ABCI iscommercially available in small quantities as a naturally derived enzyme(Seikagaku Corporation) and can be made by a recombinant productionsystem to have essentially the same activity as the enzyme purified fromProteus vulgaris. Genomic DNA can be isolated from Proteus vulgarisusing DNeasy Tissue kit (Qiagen). PCR primers can be synthesized with anNdeI restriction site at the 5′ end and a BamHI site at the 3′ endhaving sequences 5′-CAT ATG GCC ACC AGC MT CCT GCA TTT G-3′(F2) (SEQ IDNO: 95) and 5′-GGA TCC TCA AGG GAG TGG CGA GAG-3′(R) (SEQ ID NO: 96)respectively, to synthesize the mature protein. The 3.0 kb PCR productscan be ligated into pCR 2.1 vector (TOPO cloning kit, Invitrogen) andtransformed into DH5a competent cells (Invitrogen). Plasmid DNA can beisolated from a number of clones screened by digestion with EcoRIrestriction enzyme. The integrity of a gene prepared in this way can beconfirmed by repeated DNA sequencing.

The chondroitinase ABCI sequence can cloned into a PET vector (Novogen)for expression in E. Coli After induction of gene expression with IPTGthe bacteria can lysed by sonication with the concomitant extraction ofchondroitinase ABCI with Triton X-114/PBS. The inventors discovered thatthe majority of recombinant chondroitinase ABCI was found in thecytosolic fraction of the bacterial cell lysate that enabled thedevelopment of a chondroitinase ABCI purification protocol that yieldsan enzyme with high activity at high yields. The protocol includescation-exchange chromatography as a capture step and gel filtration as apolishing step. After these steps chondroitinase ABCI reaches a purityof ˜95%. Anion exchange membrane filtration (Intercept Q, Millipore) canbe used for endotoxin and host DNA removal. This step is expected toremove approximately 75% of the endotoxin. Following filtration,chondroitinase ABCI can be dialyzed into volatile buffer, pH 8.0 andlyophilized to dryness. The final product is stable at −70° C. for longterm storage. The purified cABCI is a highly basic protein with pI-9.5as determined by IEF-PAGE analysis of the samples from the crude celllysate.

A variety of analytical methods can be used to compare the enzymaticactivity of the recombinant version of chondroitinase ABCI to that of acommercially available form of the enzyme (Seikagaku Corporation)purified from Proteus vulgaris. The methods may be adapted to evaluatethe activity of fusion proteins including proteoglycan degradingpolypeptides like chondroitinase. Specific activity measurements wereobtained using an accepted spectrophotometric assay that measures thechange in absorbance due to the production of reaction products from thedegradation of proteoglycans. The recombinant form of chondroitinaseABCI had approximately 25% higher specific activity than the Seikagakuchondroitinase ABCI. Size exclusion chromatography can be used tocompare the hydrodynamic properties the enzymes. The elution profilesfor recombinant enzyme was identical to that of the naturally derivedenzyme.

A form of zymography can used to further characterize the enzyme and maybe adapted for characterization of the fusion proteins. Polyacrylamidegels can be polymerized in the presence of aggrecan, a substrate forchondroitinase ABCI. Enzyme samples may be resolved on theaggrecan-impregnated gels by electrophoresis in the presence of SDS. Thegels can then be subjected to a renaturation step wherein the SDS wasextracted and the enzymes allowed to refold. Enzyme refolds and regainsactivity then digests aggrecan within the gel and the resulting loss ofcarbohydrate in that region of the gel can be visualized by acarbohydrate-specific stain. A similar loss of carbohydrate in the gelwould be expected for active forms of a fusion protein including aproteoglycan degrading polypeptide portion. In the case of recombinantChondroitinase ABCI, its activity can be visualized as a clear spot inthe zymogram. The zymography results were consistent with thespectrophotometric analysis that demonstrates that the recombinant formof chondroitinase ABCI has the same or greater specific activity as thenaturally occurring form.

HPLC methods may be used for detecting the four and six sulphateddisaccharides (A4DS and A6DS, respectively) liberated as a result ofchondroitinase ABCI digestion of CSPG. The two disaccharides can beeffectively resolved by anion exchange chromatography. The HPLC assayhas been validated by showing that the quantitation of A4DS and A6DSfrom chromatograms yields a linear relationship to the amounts injectedinto the HPLC. Production of A4DS and A6DS from CSPG digestion isdirectly related to the amount of chondroitinase specific activity asdetermined by the spectrophotometric assay described above. This assaymay be used as a sensitive and accurate means to independentlyquantitate A4DS and A6DS released by chondroitinase digestion of avariety of substrates and may also be used to determine the activity ofchondroitinase polypeptides in a fusion protein.

Another functional assay that can be performed to characterizeproteoglycan polypeptide activity is where dorsal root ganglian (DRG)neurons are plated on aggrecan or aggrecan treated with a proteoglycanlike chondroitinase ABCI. It is expected that neurons plated on aggrecanwill failed to adhere to the plate and extend axons. In contrast,neurons plated on aggrecan treated with a proteoglycan degradingpolypeptide like chondroitinase ABCI in a composition or as part of afusion polypeptide would be expected to adhere to the surface and extendaxons. The extensive axon growth, which is observed for chondroitinaseABCI is believed to be due to the digestion of the carbohydrates on theaggrecan core protein which creates a more permissive substrate for axongrowth.

The rat contusion model of SCI is a clinically relevant model and may beused to evaluate the efficacy of fusion proteins and other compositionsof the present invention for promoting axon regeneration. With acontusion SCI, cells are destroyed, hemorrhage ensues and inflammationbegins. Destroyed cells are removed by macrophages and a reactivegliosis begins. A cystic cavity is formed and the gliosis matures into aglial scar. Myelin in the area is destroyed and many local neurons thatare not destroyed are left in a demyelinated state.

The forceps compression model of SCI is a contusion model developed andcharacterized. This model has been validated and results in injuriesthat are very similar to the more widely used impactor models. The modelcan involves a forceps compression at vertebral level T9/T10. Theforceps compress the cord to a width of 0.9, 1.3 or 1.7 mm for 15seconds. These three levels of compression allow a severe, mild ormoderate injury. This model has been validated using the open fieldlocomotor testing and the Basso, Bresnahan and Beattie (BBB) scoringsystem. It was also characterized histologically. Behavioral testing andBBB scoring demonstrated that the forceps produce a highly reproducibleinjury, with recovery similar to that seen with impactor models. Thesetesting and scoring data demonstrate that the forceps compression modelis comparable to other contusion models and sufficiently reproducible tobe used as an experimental model of SCI and may be used for theevaluation of compositions of the present invention.

Tissue from forceps compression injured animals can be processedhistologically to examine white matter sparing, glial scar and cystformation. As with other contusion SCI models, a central cyst is formedafter injury, with a size that increases with increased injury severity(decrease forceps gap). Around the cyst a glial scar forms that ischaracterized by astrogliosis (GFAP), macrophage activation and myelinwasting.

Various aspects of the present invention will be illustrated withreference to the following non-limiting examples.

Example 1

This example illustrates how a proteoglycan degrading molecule may beadministered, studied, and demonstrated to show a functional improvementin animals having a model contusion injury.

The forceps compression model of SCI is a contusion model developed andcharacterized. This model has been validated and results in injuriesthat are very similar to the more widely used impactor models. The modelcan involves a forceps compression at vertebral level T9/T10. Theforceps compress the cord to a width of 0.9, 1.3 or 1.7 mm for 15seconds. These three levels of compression allow a severe, mild ormoderate injury.

Rats were injured with the forceps compression model at vertebralT9/T10/ At the time of surgery an intrathecal catheter was placed fordelivery of chondroitinase. Animals were treated every day for one weekand then on alternating days for one week with 0.06 U/dosechondroitinase ABC I (Seikagaku), penicillinase or artificialcerebrospinal fluid (aCSF). Doses and controls were derived fromBradbury et al., 2002. Behavior was assessed using open-field locomotortesting and the BBB scoring system at day 2 and then weekly post injuryfor ten weeks.

FIG. 5A shown are mean BBB scores for animals treated withchondroitinase ABC I, penicillinase and aCSF. Rats treated with aCSF orpenicillinase recovered to a mean BBB score of about 4. Rats treatedwith chondroitinase recovered to a mean BBB score of about 8. Multipleanimals recovered to scores above 10, indicating supra-spinal input. Thechondroitinase scores were significantly different from both controlgroups by ANOVA and post hoc Tukey.

Tissue from these animals was processed immunohistochemically for glialfibrillary acidic protein (GFAP) to assess general scar architecture.Tissue was also stained with a Weil stain and a silver degenerationstain to assess myelin and neuron degeneration, respectively.Interestingly no obvious differences in these parameters were notedbetween experimental and control treated tissues.

In FIG. 5B the large lesion comprising several segments and the sparingin the ventral cord. Weil staining revealed extensive demyelination inboth the treated and untreated animals. The GFAP images (middle set)demonstrate the extent of the scar that is formed following forcepsinjury. The bottom amino cupric silver degeneration stain demonstratesthe vast neural degeneration extending both rostrally and caudally afterinjury. Again, no obvious differences were noted between chondroitinasetreated and control tissues.

Additional preliminary experiments have been performed at moderateinjury levels and significant improvement in open-field locomotoractivity was observed with chondroitinase treatment. Animals receivingchondroitinase ABCI recovered to a mean BBB score of 9.1 at ten weeksfollowing injury, compared with 7.1 for the penicillinase controls. Theconsistency of the data (SEM's of 0.6 and 0.3, respectively) and theregion of scores on the BBB scale make this 2 point change not onlystatistically significant, but also clinically meaningful. A score of 9indicates plantar placement with weight support or frequent toconsistent weight support with dorsal stepping while a score of 7indicates movement of each joint in hind limb but with no weight supportand no consistent sweeping of limbs. An examination of the individualanimal scores at ten weeks shows that 6 of 12 animals in thechondroitinase group recovered to scores of 9 or above, while only oneof 12 animals in the penicillinase group recovered to a score of 9. Oneanimal from each group was removed from analysis because its scorefailed to ever rise above 2.5, indicating an injury severity outside themodel norms. FIG. 5C and FIG. 5D includes a scatter plot of scores at 10weeks for each animal in the penicillinase and chondroitinase treatmentgroups for the moderate injury. Group means are shown below.

The results show in this well controlled study, that chondroitinaseimproves open-field locomotor function in Rats that were injured withthe forceps compression model at vertebral T9/T10. Animals recovered tomean BBB scores of 9.7 and 9.9 for the penicillinase and chondroitinasegroups, respectively. This study demonstrates a significant effect atsevere and moderate injury levels, but not mild injury levels. Nosignificant differences were noted between any groups in the mild injurystudy (1.3 mm forceps). It is unclear if chondroitinase is not effectivewith mild injury, if chondroitinase effected changes not adequatelyassayed by the open-field locomotor testing such as stride length, pawplacement, sensory or autonomic functions. Preliminary analysis ofhistology from animals in this study confirmed placement of thecatheters and injuries in each animal. Ongoing experiments sacrificinganimals at time points after injury with and without chondroitinasetreatment will characterize the CSPG content of the scar and the effectsof chondroitinase digestion on stub-antigen expression. Futureexperiments will expand the battery of behavior tests and examine thecord for evidence of regeneration with tract tracing.

Two acute toxicity studies were conducted in rats. The first was anintravenous (IV) study wherein rats were injected with 0, 0.2, 0.775 or7.775 mg/kg chondroitinase ABCI. In the second study, intrathecal (IT)catheters were placed over the spinal cords using the same methodsemployed in the SCI animal studies and 0.06, 0.6 and 6.0 units ofchondroitinase ABCI was delivered through the IT catheters. These doseswere 1, 10, and 100-fold greater than the estimated local concentrationsof chondroitinase ABCI achieved with IT catheters in the SCIexperiments. Animals were monitored for pain and distress and bodyweights were acquired daily. No overt reactions were observed during orimmediately after chondroitinase ABCI dosing. No swelling, inflammation,bruising or necrosis was noted at the injection site for the IVexperiment. No changes in body temperature were observed in animalstreated via the IT route. No alterations in feeding, grooming orvocalizations were noted. Animals were assessed for motor behavior in anopen pool. No abnormalities were noted by the animal care staff orbehavioral specialists. Animals displayed no signs of joint tendernessor swelling. There were no significant differences in weight changebetween the treatment groups. The results demonstrate thatchondroitinase ABCI treatment is not associated with acute toxicityusing IV and IT doses substantially greater than the efficacious ITdoses.

Example 2

This example describes the preparation of Nogo-receptor agonist, L1neural cell adhesion protein, and GGF2 polypeptide domains which can beused for compositions and fusion proteins of the present invention.

A soluble portion of the human Nogo receptor spanning amino acids 27 to311 (NgR₂₇₋₃₁₁) was selected as it has been shown to inhibit the bindingof Nogo66, MAG, and MOG to membrane-bound NgR. Primers were designedflanking this region, and RT-PCR was performed using human hippocampalRNA (BD Biosciences). The 1.05 kb region was successfully amplified andpurified.

L1 was prepared through a CHO cell line from the laboratory of Dr.Melitta Schachner that secretes human L1 as a fusion protein with humanFc (L1-Fc). The cells were grown in roller bottles and then L1-Fc waspurified from the conditioned media using protein A affinity columnchromatography. The purity of L1-Fc was assessed by SDS-PAGE and asingle band at the appropriate molecular weight was observed. Thebiological activity of L1-Fc was confirmed using a neurite outgrowthassay. Tissue culture plates were coated with either poly L-Lysine orL1-Fc and then cerebellar granule cells from postnatal day 10 rats wereisolated and placed into culture on the substrates. It was observed thatneurons plated on the L1-Fc substrate exhibited a substantial number oflong neuritis compared to the polylysine substrate controls. Theseresults demonstrate that the L1-Fc produced is biologically active inpromoting neurite outgrowth. This neurite outgrowth assay will be usedto assess the biological activity related to the L1 portion of thechondroitinase ABCI fusion protein.

A CHO cell line that secretes a soluble form of full length GGF2glycoprotein was obtained from CeNes. Extensive optimization of mediaand purification methods was completed to obtain essentially pure andbiologically active GGF2. A number of analytical methods were developedto characterize the GGF2 including SDS-PAGE, isoelectric focusing,peptide mapping and carbohydrate analysis. For example an SDS-PAGE gelof reduced and non-reduced GGF2; the isolate is essentially free ofcontaminating proteins and shows the expected molecular weight andmonomeric structure. The biological activity of GGF2 was assayed using aprimary rat Schwann cell proliferation method and the expected effectwas reproducibly obtained with four independent batches of GGF2. Anotherfunctional assay was developed that measures the phosphorylation of Aktkinase, a downstream cell signaling component of the erbB receptorpathway. It was observed that there is a dose dependent phosphorylationof Akt kinase

Example 3

This example illustrates the manipulation of chondroitinase ABCI forconstruction of chimeras.

Chondroitinase ABCI was cloned from P. vulgans and expressed in E. coli.Surprisingly repeated attempts to create N-terminal fusion proteins werenot successful because the N-Terminal fusion portion was cleaved fromchondroitinase ABCI during synthesis.

However, catalytically active deletion mutants with N-terminal Hisfusion tags designated ABC I-NΔ2O, ABC I-NΔ4O, and ABCI-NΔ6O wereprepared by deleting 20, 40 and 60 amino acids respectively from theN-terminus of the mature ABCI protein. Unlike the full lengthchondroitinase ABCI, the N-terminal deletion mutants are capable ofsynthesizing a 6xHis tag as an N-terminal fusion protein. It was alsoobserved that deletion of 80 amino acids from the C-terminal end formeda mutant with proteoglycan degrading activity of chondroitinase ABCI astested in a zymography assay.

Fusion proteins with NgR₂₇₋₃₁₁ or L1 with a proteoglycan degradingmolecule such as chondroitinase ABCI will utilize mammalian expressionand can be performed in CHO cells. The cDNA for chondroitinase ABCI hasbeen cloned in pSECTag vector (Invitrogen) in the proper reading frame.A CHO cell line having secretary chondroitinase ABCI can be developedand the conditioned medium can be tested for catalytic activity byzymography assay to confirm that chondroitinase ABCI expressed inmammalian cells is functional. A mammalian cell codon optimized versionof chondroitinase ABCI can be synthesized by methods known in the artfor use for CHO cell expression.

GGF2 is a spliced variant of the NRG1 gene expressed in brain and spinalcord of adult humans. It is a glycosylated protein of molecular massbetween 66-90 kDa. The inventors have discovered that recombinant GGF2expressed in CHO cells is highly glycosylated and promotes Schwann cellproliferation in vitro and further that an EGF-like domain of NRG1expressed in E. coli is fully functional in promoting myocyteproliferation and survival.

The inventors have expressed fragments of GGF2 in E. coli as described.The specific GGF2 domain responsible for Schwann cell proliferation andthereby remyelination can be determined. If the Ig and EGF domainstogether or separately show biological activity in vitro, then they canbe used to form chimeric fusion proteins.

Cloning and expression of NgR₂₇₋₃₁₁ in CHO cells. An NgR fragmentcorresponding to residues 1-359 was isolated by RT-PCR from humanhippocampus poly K RNA (BD Biosciences) and its structure can beconfirmed by DNA sequencing. The gene fragment corresponding to residues27-311 can be cloned from the larger fragment and then subcloned inpSECTag vector (Invitrogen) in the proper reading frame to express thefragment as a secretary protein in CHO cells. The plasmid DNA containingthe NgR₂₇₋₃₁₁ gene can be transfected in CHO cells and the cell lineproducing NgR₂₇₋₃₁₁ can be selected under the selection pressure ofhygromycine B. An NgR₂₇₋₃₁₁-ABCI chimera expression plasmid can beconstructed for expression in a CHO cell expression system using methodsknown in the art.

Example 4

This example illustrates methods which may be used to purify and isolateexpressed chondroitinase ABCI, and GGF2 domains expressed in E. coli.These method may be applied to purification of chimeric fusion proteinsof the present invention.

An efficient E. coli recombinant expression system and a purificationprocess for chondroitinase ABCI have been developed by the inventorsthat could be applied for the purification of chondroitinase ABCIchimeric derivatives.

For example, the expression of various GGF2 domains in thechondroitinase ABCI expression host, E. coli has been performed and thefollowing peptides have been tested: aa250-402, aa250-422 and aa350-402.These expressed peptides were found in the soluble fraction of the E.coli lysates. It is reasonable to expect that final chimeric products ofthese peptides with chondroitinase ABCI in E. coli will also be soluble.In addition, it is expected that the charge characteristics of thechimeric products when compared to the recombinant chondroitinase ABCIwill be similar. Thus, the theoretical isoelectric point (p1) values forthe GGF2 peptides are 9.3 for aa250-402, 9.18 for aa250-422 and 7.55 foraa350-402. Fusing the first two peptides with chondroitinase ABCI isexpected to result in chimeric proteins with p1 values still above 9. Inthis case SP chromatography is expected to perform well as the capturingstep. The smallest GGF2 peptide, aa 350-402, will reduce the p1 of thefinal chondroitinase ABCI chimera to approximately 8.4. This change mayrequire optimization of the capture step conditions.

The chimeric products of chondroitinase ABCI with NgR₂₇₋₃₁₁ and L1peptides can be expressed in a CHO cell system. Prior to purification ofthe expressed chimeric proteins, the growth conditions for the cell lineproducing either the NgR₂₇₋₃₁₁ or L1 chimeric proteins will be optimizedin serum-free medium. A detailed media optimization study can beperformed to determine the highest production conditions. Scale-upvolume can be decided based on the rate of production of the chimericproteins (pg/cell/day). Conditioned media from the variouschimera-producing cell lines can be collected and subjected totangential flow filtration. Ion-exchange chromatography can be used forcapturing the secreted proteins from conditioned media, gel filtrationchromatography can be used as a polishing purification step and thenanion-exchange membrane filtration can be used for endotoxin and DNAremoval. At each step the efficiency of the purification will beanalyzed by SDS-PAGE, and spectrophotometric quantitation of proteinconcentration.

Example 5

This prophetic example describes in vitro assessment of chimerabiological activity: Each chimera can be assayed for chondroitinaseenzymatic activity and the specific biological activity of each fusionpartner.

The first step in analysis may employ conventional protein biochemicalmethodologies to confirm the fidelity of gene expression. These includeSDSPAGE, lEE, mass spectrometry and size exclusion chromatography.

Chondroitinase chimera specific activity can be determined using astandard and uniformly accepted spectrophotometric assay. The productionof reaction products from the catalytic activity of a chondroitinasechimeric polypeptide can be determined by a measurement of theabsorbance of the product at a wavelength of 232 nm. A typical reactionmixture consists of 120 microliters of reaction mixture (40 mM Tris, pH8.0, 40 mM NaAcetate, 0.002% casein) combined with substrate (5microliters of 50 mM chondroitin C) and 1.5 microliters ofchondroitinase ABCI chimeric fusion polypeptide or test sample. Thechange in absorbance units is the initial rate which can be converted toa unit activity measurement.

Disaccharide HPLC assay: The catalytic activity of chondroitinasechimeric polypeptide on a CSPG substrate is expected to releases twospecies of sulphated disaccharides including α-Δ AUA-[1→3]-GaINAc-4S (Δ4DS) and α-Δ AUA-[1→3]-GaINAc-6S (Δ 6DS). These species can be resolvedby HPLC and the quantitation from the resulting chromatograms is asensitive and accurate measure of chondroitinase activity. To performthe assay, samples from chondroitinase digestion reactions carried outwith wild-type chondroitinase and chondroitinase fusion proteins can beclarified by centrifugation and then subjected to a validated anionexchange HPLC method as follows. A Dionex CarboPac PA-10 analyticalcolumn (4×250 mm) fitted with a Dionex CarboPac PA-10 (4×50 mm) guardcolumn can be used with a mobile phase consisting of a gradient of waterat pH 3.5 (Buffer A) and 2M NaCl, at pH 3.5 (Buffer B). Detection may beset at a wavelength of 232 nm. A flow rate of 1 mL/minute and a 45minute continuous gradient of 100% A to 100% B affords acceptableresolution of Δ4DS and Δ 6DS. Standard curves can be generated usingknown amounts of Δ4DS and Δ6DS.

Zymography allows resolution of proteins by molecular weight with aconcomitant assessment of chondroitinase activity. A 10% polyacrylamidegel may be polymerized in the presence of 85 pg/ml of aggrecan. Samplescan be boiled in an SDS-loading buffer and then the analytes can beresolved by electrophoresis. After separation the gel can be incubatedfor 1 hour at room temperature in 2.5% Triton X100 then 16 hours at 37°C. in fresh 2.5% Triton X-100. During these incubations, the SOS can beextracted from the gel and the chondroitinase refolds and digests theaggrecan in its immediate vicinity. After the refolding process the gelcan be stained for carbohydrate: The gel can be first incubated in 0.2%Cetylpyridinium for 90 minutes at room temperature and then transferredinto 0.2% Toludine Blue in 49:50:1 H20, Ethanol, and Acetic Acid for 30minutes. The gel can then be fully destained. Following destaining thegel can be incubated overnight in a 50 microgram/ml solution ofStains-All (Sigma) in 50% ethanol. Chondroitinase activity can bedetected as a clear spot in the gel that is coincident with themolecular weight of the enzyme. The size of the clearing has been shownto be almost linearly related to Unit activity.

The NgR₂₇₋₃₁₁-chondroitinase chimera can be assessed for the activity ofthe decoy Nogo receptor. The assay can be used to measures the collapseof growth cones: Dorsal root ganglia (DRG) are dissected from postnatalday 1 (P1) Sprague Dawley rat pups and dissociated in 200 U/mlcollagenase I (Worthington) and 2.5 U/ml dispase (Boehringer/Roche) 2times for 30 min. at 37° C. Enzymes are removed and DNAse (0.5 mg/ml)can be added to the ganglia. Trituration may be done with a pipette tipattached to a 1000 μl Pipetman. The resulting cell suspension can befiltered through a 40 micron cell filter and centrifuged at 70×g for 5min. Cells can be re-suspended in DMEM/10% FBS and pre-plated for 2hours on a non-coated tissue culture plate (100 mm diameter).Non-adherent neurons are removed and plated at 10,000 cells/well in aPoly-lysine/laminin-coated 24 well plate in serum-free Neurobasal/B27with 50 ng/ml NGF. After 20 to 24 hours, MAG or Nogo66 can be added atvarying concentrations for 1 hour at 37° C. to induce growth conecollapse. NgR₂₇₋₃₁₁-chondroitinase chimera can be added at variousconcentrations to compete with MAG and Nogo66 and thus protect theneurons from growth cone collapse. Cultures may be fixed by adding anequal volume of pre-warmed 8% paraformaldehyde/0.6 M sucrose to themedium for 20 min. while the cells are kept on a 37° C. hot plate.Growth cones may be labeled with AlexaS68 phalloidin (Molecular Probes).Briefly, cells can be permeabilized with 0.1% Triton-X 100 for 5 min. atRT, blocked for 20 min. in 1% BSA in PBS and incubated in phalloidin,diluted 1:40 in 1% BSA, in PBS for 20 mm at RT. Cells can be washed inPBS and mounted in Fluorescent Mounting Medium (DAKO). The percentage ofcollapsed growth cones is determined by analyzing a minimum of 100growth cones per well under a 40× objective.

The biological activity L1 fusion proteins may be determined using astandardized neurite outgrowth assay. After etching a 25 mm circle inthe center of a tissue culture-treated 35 mm dish, 1 ml of 10 μg/mlpoly-lysine can be added to each etched circle and incubated at 37° C.for 60 minutes. The poly-lysine provides a negative control for neuriteoutgrowth. Etched circles may be washed 2 times with Hank's balancedsalt solution plus calcium and magnesium (HBSS⁺⁺) after the last rinse;1.2 μl of L1-Fc (0.6 μM, 0.3 μM, 0.15 μM, 0.075 μM) is spotted onto thepetri dishes to serve as a positive control and various concentrationsof the L1-chondroitinase fusion protein can be applied to the plates astest samples. The plates can be incubated for 1 hour at roomtemperature. The spots are lightly aspirated, so as not to dry them out,and immediately washed 2 times with 1 ml HBSS⁺⁺ and then 1 ml of 1% BSAin PBS is added to each etched circle. After about 15 minutes at roomtemperature the etched circles can be washed 2 times with HBSS⁺⁺, andonce with bioassay medium (NeuralBasal (Gibco)+B27 supplements(Gibco)+L-glutamine+L-glutamic acid, penicillin and streptomycin (100U/ml)+1% fetal bovine serum which will remain on the petri dishes untilthe time of the assay. To provide neurons to plate on the substratesCerebellar granule cells from postnatal day (PND) 9 or 10 are harvested.The brain is removed from the cranium of 1 pup, and the cerebellumseparated from the rest of the tissue. Meninges is removed, and thecerebellum is placed in ice cold HBSS⁺⁺. The tissue is minced andtrypsinized using 0.25% trypsin for 15 minutes at 37° C. Trypsin actionis inhibited by adding 0.5 mg/ml soybean trypsin inhibitor (Gibco). Thetissue is rinsed with HBSS⁺⁺ and triturated using a flame narrowedPasteur pipette coated with EBS. Dissociated cells are pelleted at 500×gfor 3 minutes, the supernatant is decanted and the cells are resuspendedin 2 ml of growth medium. After lightly triturating, the cell suspensionis carefully layered on top of a 3.5% BSA (in PBS) cushion, and spun at500×g for 3 minutes. Supernatant is aspirated and the pellet isresuspended in 2 ml growth media. A cell count is performed and thecells are diluted to a final working concentration of 1.5×105 cell/ml. A300 μl aliquot of diluted cells is added to the center of each plate,resulting in the even distribution of 6×104 cells across the entiresurface area of the etched circle. The cells are allowed to grow for 16hours at 37° C. in a humidified environment supplemented with 5% CO2.The next day, plates are removed from the 37° C. incubator, cells arefixed with 4% paraformaldehyde and the outgrowth of neurites is recordedby photomicroscopy.

Biological activity assays for GGF2 may be performed using Schwann cellproliferation. Sciatic nerves are dissected from three day old SpragueDawley rat pups and dissociated in L-15 medium (Invitrogen) containing0.25% trypsin and 0.03% collagenase type I (SIGMA) for 15 minutes at 37°C. Nerves are centrifuged at 400×g for 5 minutes and dissociation mediumis replaced by DMEM/10% FBS. Nerves are triturated using a 10 ml syringewith a 21 g needle and subsequently a 23 g needle. The cell suspensionis filtered through a 40 μm mesh placed over the opening of a 50 mlconical tube. Cells can be centrifuged at 400×g for 5 min, plated in apoly-D-lysine (PDL) coated T-75 flask at a density of approximately 5million cells in 15 ml of DMEM/10% FBS/PenStrep and incubated in a 37°C. incubator regulated with 10% CO₂. After a 24 hour incubation thecells can be washed twice with DMEM/10% FBS and re-fed with fibroblastinhibition medium consisting of DMEM/10% FBS and 10 μl/ml of 1 mMcytosine arabinoside (Ara-C). After an incubation time of 2 to 3 daysthe fibroblast inhibition medium is replaced with Schwann cell growthmedium (DMEM/10% FBS/150 ng/ml of GGF2/5 μM forskolin). Cells can beexpanded and aliquots of 2×106 cells/ml are frozen in DMEM/10% DMSO, 54%FBS in liquid nitrogen. At the time of use, Schwann cells are thawed andplated at a density of about 16,000 cells/well in a PDL-coated 96 welltissue culture plate in DMEM/5% FBS. After about 24 hours GGF2 is addedin serial dilution ranging from 100 ng/ml to 0.78 ng/ml containing 5 μMforskolin to establish a standard curve. Samples of GGF2 fusion proteinscan also be added in serial dilution together with 5 μM forskolin. BrdUis added at 10 μM. Cells are incubated for 48 hours in a 10% CO₂incubator. A BrdU ELISA kit from Roche Applied Science can be used (Cat.No. 1 647 229) to detect Schwann cell proliferation. Medium is pouredout of the plate and plate is tapped on tissue paper. A 200 μl/wellaliquot of Fix/Denat is added and incubated for 30 min at 15-25° C. A100 μl/well aliquot of anti-BrdU-POD working solution (lyophilizedantibody is dissolved in 1.1 ml double distilled water and diluted 1:100with antibody dilution solution) can be added and incubated for 90 minat room temperature. Wells may be washed 3 times with 200-300 μl/well ofwashing solution. A 100 μl aliquot of substrate solution is added andincubated for 15 minutes or until color development is sufficient forphotometric detection. The Plates are read on a SpectraMax plate readerat 450 nm either before addition of stop solution at 370 nm or afteraddition of 50 ml 1N sulfuric acid to each well.

Akt [pS473] ELISA: C6 glioma cells, obtained from ATCC, are grown inDMEM/10% FBS in a T-75 flask to confluence. After trypsinization, cellsare plated in a 24 well plate at a density of 500,000 cells/well in 0.5ml of medium. One day after plating the cells are treated with GGF2(batch: Glu from Lonza Biologics) at serial dilutions ranging from 0.78to 100 ng/ml for 30 minutes at 37° C. to establish a standard curve. Asa negative control, wortmannin, a PI3-kinase inhibitor, is added tocells at 10 nnM for 30 minutes before the addition of GGF2. Samples ofGGF2 fusion proteins will also be added in serial dilution. The cellsare washed with PBS and then extracted with 100 μl of cell extractionbuffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF,20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% SDS,0.5% deoxycholate, 1 mM PMSF, protease inhibitor cocktail (Pierce)).Cell extracts are kept at −80° C. until further use. An ELISA kit fromBiosource (Cat. #KHOO1 II) can be used to measure phosphorylated Aktkinase levels. Briefly, 100 μl of a 1:50 dilution of samples and aserial dilution of Akt [pS473] standards are loaded into wellspre-coated with anti-Akt antibody for 2 h at room temperature (RT) orovernight at 4° C. Wells are washed and anti-Akt [pS473] antibody isadded and incubated for 1 hour. After washing, HRP-conjugatedanti-rabbit antiserum is added to wells and incubated for 30 min. Afterwashing, TMB chromogen is added to wells and incubated for 30 min at RTbefore the reaction is stopped with stop solution. The plate is read ona SpectraMax plate reader at 450 nm. The optical densities are plottedagainst the Akt [pS473] standards and the concentration of pAkt in theC6 samples are deduced from the standard curve.

Example 6

This example illustrates construction of a fusion protein ofchondroitinase polypeptide and a TAT cellular transduction peptide.

The gene sequence encoding a chondroitinase enzyme can be functionallylinked to the protein transduction domain from HIV called the TATPeptide (SEQ ID NO: 61). The resultant chimeric gene TAT-chondroitinaseABCI fusion DNA construct is shown in FIG. 1. It was observed thatduring bacterial expression of this construct, the TAT peptide wasremoved from the chondroitinase enzyme at some processing point duringthe bacterial growth. The removal of n-terminal linked peptides was alsoobserved during expression of an n-terminal histidine-taggedchondroitinase ABCI enzyme.

Deletion mutants of the chondroitinase ABCI enzyme were generated wherethe mutant was missing a certain number of amino acids from then-terminal portion of the polypeptide but maintained it proteoglycandegrading activity. It was observed that these n-terminal deletionmaintained a histidine-tag that was attached to the n-terminus. It isexpected that various TAT-deletion mutant chondroitinase ABCI fusion DNAconstruct can be expressed without removal of the TAT polypeptide duringexpression. For example, the TAT peptide may be fused at the N-terminusof deletion mutants like ABCI-NΔ20 or ABCI-NΔ60 deletion mutant. Withoutwishing to be bound by theory, the inventors believe that the nativeproteoglycan degrading enzyme contains a signal sequence that isattached to the n-terminus. This signal sequence is removed during thenatural production in bacteria and in production of the cloned enzyme inE. coli. It is thought that some signal within the n-terminal aminoacids instructs the bacteria to remove anything attached to this end.

A DNA construct with the TAT-peptide attached to the N-terminus of oneof the chondroitinase deletion mutants can be made and Western blot andprotein gel showing this expressed protein and activity.

Example 7

This example illustrates the diffusion of molecules into cells andtissue using a proteoglycan degrading composition.

A brain from an adult Sprague Dawley rat was removed from the skull andquartered into Right frontal, left frontal, right rear and left rearsections, corresponding roughly to the frontal (front) andoccipito-parietal (rear) lobes. (A) Right frontal quarter was placed inartificial cerebrospinal fluid (Catalog #59-7316; Harvard Apparatus,Holliston, Mass.) containing the beta-galactosidase enzyme (Catalog #, G5160; Sigma, St. Louis, Mo.) and chondroitinase ABC I at 0.5 U/ml(Catalog# C 3667; Sigma, St. Louis, Mo.) for 2 hours at 37° C. Brainquarter was rinsed several times in phosphate buffered saline (PBS) andthen processed with a Beta-Gal staining kit (Catalog # Gal-S; Sigma, St.Louis, Mo.). The substrate-enzyme reaction (blue product) was allowed todevelop for 1 hour, and the brain was rinsed several times in PBS andslabs from the middle of each brain block cut using parallel straightrazors. (B) Left frontal quarter was placed in artificial cerebrospinalfluid (Catalog #59-7316; Harvard Apparatus, Holliston, Mass.) containingthe beta-galactosidase enzyme (Catalog #, G 5160; Sigma, St. Louis, Mo.)for 2 hours at 37° C. Brain quarter was rinsed several times inphosphate buffered saline (PBS) and then processed with a Beta-Galstaining kit (Catalog # Gal-S; Sigma, St. Louis, Mo.). Thesubstrate-enzyme reaction (blue product) was allowed to develop for 1hour, and the brain was rinsed several times in PBS and slabs from themiddle of each brain block cut using parallel straight razors. (C) Rightfrontal quarter was placed in artificial cerebrospinal fluid (Catalog#59-7316; Harvard Apparatus, Holliston, Mass.) containing thebeta-galactosidase enzyme (Catalog #, G 5160; Sigma, St. Louis, Mo.) andchondroitinase ABC I at 0.5 U/mi (Catalog# C 3667; Sigma, St. Louis,Mo.) for 2 hours at 37° C. Brain quarter was rinsed several times inphosphate buffered saline (PBS) and then processed with a Beta-Galstaining kit (Catalog # Gal-S; Sigma, St. Louis, Mo.). Thesubstrate-enzyme reaction (blue product) was allowed to develop for 1hour, and the brain was rinsed several times in PBS and slabs from themiddle of each brain block cut using parallel straight razors. (D) Leftrear quarter was placed in artificial cerebrospinal fluid (Catalog#59-7316; Harvard Apparatus, Holliston, Mass.) containing thebeta-galactosidase enzyme (Catalog #, G 5160; Sigma, St. Louis, Mo.) for2 hours at 37° C. Brain quarter was rinsed several times in phosphatebuffered saline (PBS) and then processed with a Beta-Gal staining kit(Catalog # Gal-S; Sigma, St. Louis, Mo.). The substrate-enzyme reaction(blue product) was allowed to develop for 1 hour, and the brain wasrinsed several times in PBS and slabs from the middle of each brainblock cut using parallel straight razors. Images of brains were acquiredon a scanner and are shown in FIG. 2(I)(A-D).

Adult rat brain hemispheres were soaked in buffer alone or containing 33U/ml Chondroitinase ABCI (Acorda) for 2 hours at 37 degrees C.Hemispheres were rinsed and immediately placed in Eosin Y (Sigma) or asaturated solution of Congo Red (Sigma) in 70% ethanol. Slabs of tissuewere cut and images were acquired on a scanner. See FIG. 2(II) Eosin andFIG. 2(III) Congo red.

FIG. 2(III) saturated solution of Congo Red demonstrates greaterpenetration through the cortex of a Chondroitinase treated brainhemisphere as compared to untreated brain. Congo Red is a negativelycharged dye of 697 kDA. FIG. 2(II) Eosin Y penetration through thecortex of a Chondroitinase treated brain hemisphere looks slightly morediffuse, but penetration does not seem to be any deeper as compared tountreated brain. Eosin Y is zwitterionic, having an overall negativecharged at the low pH it was used at, and it is 692 kDa. FIG. 2(I) Lobesfrom adult rat were incubated in beta-galactosidease alone (B&D), orwith the addition of Chondroitinase ABCI (A, 0.5 U/ml or C, 0.005 U/m).

The results showed that chondroitinase treated tissue affectedpenetration of beta-galactosidase into CNS tissue. Chondroitinse haddramatic effects on the rate of Congo Red dye penetration but apparentlynot Eosin.

Example 8

This example illustrates a Chondroitinase ABCI Assay Protocol which maybe modified to measure the activity of Chondroitinase ABCI deletionmutant-fusion proteins or the activity of other proteoglycan degradingpolypeptide-fusion proteins of the present invention.

The production of reaction products from the catalytic activity of aproteoglycan degrading molecule or fusion protein is determined by ameasurement of the absorbance of the product at a wavelength of 232 nm.A typical reaction mixture consists of 120 μl of reaction mixture (40 mMTris, pH 8.0, 40 mM NaAcetate, 0.002% casein) combined with a substrate(5 IA of 50 mM chondroitin C) and 1.5 μl of chondroitinase ABCI or afusion protein of a deletion mutant of chondroitinase ABCI-TAT.Seikagaku's cABCI reference: frozen at −20° C. in 5 ml aliquots, use 1ml per reaction, 50 mM chondroitin C (MW 521) in water: frozen at −20°C. in 100 ml aliquots. Reaction mixture aliquots of about 120 μl can beprepared at 37° C. for 3 min or longer. A wavelength of 232 nm, is usedwith the spectrometer.

Knowing the extinction coefficient for the reaction product, measuringthe change in the absorbance of the reaction product at 232 nm readingover time upon addition of a known amount of the Chondroitinase ABCI orother proteoglycan degrading polypeptide-fusion proteins to the 120 μlreaction mixture with 0.002% casein and chondroitin substrate added, thespecific activity in μmol/min/mg of the proteoglycan degrading fusionprotein can be determined. Seikagaku Chondroitinase ABCI has a specificactivity under these assay conditions of about 450 μmole/min/mg.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontain within this specification.

1-45. (canceled)
 46. A composition comprising: a polypeptide comprisinga protein transduction domain and a proteoglycan degrading domain,wherein the proteoglycan degrading domain is selected from the groupconsisting of chondroitinase ABC I (SEQ ID NO: 50), chondroitinase ABCII (SEQ ID NO: 35), hyaluronidase-1 (SEQ ID NO: 38), hyaluronidase-2(SEQ ID NO: 39), hyaluronidase-3 (SEQ ID NO: 40), hyaluronidase-4 (SEQID NO: 41), PH-20 (SEQ ID NO: 42), chondroitinase B (SEQ ID NO: 37),chondroitinase AC (SEQ ID NO: 36), and a combination thereof.
 47. Thecomposition of claim 46, wherein the protein transduction domain is aTAT domain.
 48. The composition of claim 46, wherein the proteintransduction domain and the proteoglycan degrading domain are bondedtogether by a linker polypeptide domain.
 49. The composition of claim46, further comprising a pharmaceutically acceptable excipient.
 50. Thecomposition of claim 46, wherein the polypeptide comprises an amino acidsequence represented by SEQ ID NO:
 89. 51. The composition of claim 46,further comprising cells from the central nervous system.
 52. Thecomposition of claim 46, wherein the polypeptide further comprises adomain that promotes neural regeneration.
 53. The composition of claim52, wherein the domain that promotes neural regeneration is selectedfrom the group consisting of L1 (SEQ ID NO: 5), a functional variantthat is at least 80% L1, a functional deletion mutant of L1, GGF2 (SEQID NO:6), a functional variant that is at least 80% GGF2, a functionaldeletion mutant of GGF2, NgR27-311 (SEQ ID NO:4), a functional variantthat is at least 80% NgR27-311, and a functional deletion mutant ofNgR27-311.
 54. The composition of claim 52, wherein the domain thatpromotes neural regeneration is bonded to the protein transductiondomain or the proteoglycan degrading domain by a linker polypeptidedomain.
 55. The composition of claim 46, wherein the proteoglycandegrading domain chondroitinase ABC I (SEQ ID NO: 50) does not comprisea signal sequence.
 56. A method of promoting recovery of neurologicalfunction comprising: administering to a subject in need thereof acomposition comprising a polypeptide comprising a protein transductiondomain and a proteoglycan degrading domain, wherein the proteoglycandegrading domain is selected from the group consisting of chondroitinaseABC I (SEQ ID NO: 50), chondroitinase ABC II (SEQ ID NO: 35),hyaluronidase-1 (SEQ ID NO: 38), hyaluronidase-2 (SEQ ID NO: 39),hyaluronidase-3 (SEQ ID NO: 40), hyaluronidase-4 (SEQ ID NO: 41), PH-20(SEQ ID NO: 42), chondroitinase B (SEQ ID NO: 37), chondroitinase AC(SEQ ID NO: 36), and a combination thereof.
 57. The method of claim 56,wherein the protein transduction domain is a TAT domain.
 58. The methodof claim 56, wherein the protein transduction domain and theproteoglycan degrading domain are bonded together by a linkerpolypeptide domain.
 59. The method of claim 56, wherein the compositionfurther comprises a pharmaceutically acceptable excipient.
 60. Themethod of claim 56, wherein the polypeptide comprises an amino acidsequence represented by SEQ ID NO:
 89. 61. The method of claim 56,wherein the composition further comprises cells from the central nervoussystem.
 62. The method of claim 56, wherein the polypeptide furthercomprises a domain that promotes neural regeneration.
 63. The method ofclaim 62, wherein the domain that promotes neural regeneration isselected from the group consisting of L1 (SEQ ID NO: 5), a functionalvariant that is at least 80% L1, a functional deletion mutant of L1,GGF2 (SEQ ID NO: 6), a functional variant that is at least 80% GGF2, afunctional deletion mutant of GGF2, NgR27-311 (SEQ ID NO: 4), afunctional variant that is at least 80% NgR27-311, and a functionaldeletion mutant of NgR27-311.
 64. The method of claim 62, wherein thedomain that promotes neural regeneration is bonded to the proteintransduction domain or the proteoglycan degrading domain by a linkerpolypeptide domain.
 65. The method of claim 56, wherein the proteoglycandegrading domain chondroitinase ABC I (SEQ ID NO: 50) does not comprisea signal sequence.